Application of substance for detecting content of iron ions in preparation of product for diagnosis and prognosis of fever with thrombocytopenia syndrome

Abstract

The invention discloses application of a substance for detecting the content of iron ions in preparation of a product for diagnosis and prognosis of a fever with thrombocytopenia syndrome. The invention further discloses application of the substance for detecting the content of the iron ions in screening patients with the fever with thrombocytopenia syndrome, evaluating prognosis survival conditions or death risks or tissue damage degrees of the patients with the fever with thrombocytopenia syndrome and monitoring change of disease degrees of the patients with the fever with thrombocytopenia syndrome. It is found for the first time that the content of iron ions in serum can effectively assist in diagnosing the fever with thrombocytopenia syndrome, meanwhile, the method has an important assisting and guiding effect on prognosis of development of the fever with thrombocytopenia syndrome, doctors can conduct definite diagnosis conveniently, and a reasonable treatment scheme can be made according to prognosis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of a substance for detecting iron ion content in the preparation of a product for diagnosis and prognosis of fever with thrombocytopenia syndrome. Background Art

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a new tick-borne infectious disease first discovered in rural areas of China. Its pathogen, a new Bunyavirus, also known as severe fever with thrombocytopenia syndrome virus (SFTSV), was isolated and identified from the blood of patients in 2009. Cases are mainly distributed in rural areas in mountainous and hilly areas. The cases are highly sporadic, but relatively concentrated in terms of geographical distribution. The symptoms caused by the virus mainly include fever, thrombocytopenia and leukopenia, lymphadenopathy, gastrointestinal discomfort and muscle aches. Most patients experience only mild or moderate illness after infection, but some patients become critically ill and develop disseminated intravascular coagulation (DIC) and multiple organ failure, eventually dying. In addition to thrombocytopenia and leukocytopenia, abnormal laboratory test results also showed elevated levels of serum alanine aminotransferase, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase. The mortality rate was 10.5% (95% confidence interval [9.8-11.2%]).

[0003] The new Bunyavirus can be transmitted by a variety of ticks, mainly the long-horned blood tick. People who go out to farm, travel, and live in hilly or forested areas are at high risk, and the incidence rate gradually increases with age. At present, there have been cases of human-to-human transmission of the new Bunyavirus infection. It can be transmitted through contact with the patient's blood or body fluid contaminants, and there is a high risk of infection for medical staff, bedside companions, or funeral workers. The incubation period of the new Bunyavirus infection in humans is not very clear, it may be 1-2 weeks. In cases of human-to-human transmission, the incubation period is mostly 6 to 9 days. Summary of the invention

[0004] One object of the present invention is to provide a new use of a substance for detecting iron ion content.

[0005] The present invention provides the use of a substance for detecting iron ion content in any one of the following (a1)-(a7):

[0006] (a1) Preparation of products for diagnosis or auxiliary diagnosis of fever with thrombocytopenia syndrome;

[0007] (a2) Preparation of products for screening or assisting in screening patients with HFTS (or for screening or assisting in screening patients with HFTS who have a poor prognosis or for screening or assisting in screening patients with HFTS who have a good prognosis);

[0008] (a3) Preparation of products that can differentiate or assist in differentiating patients with HFTS who have a poor prognosis from patients with HFTS who have a good prognosis;

[0009] (a4) Preparation of products for evaluating or assisting in evaluating the prognosis and survival of patients with fever and thrombocytopenia syndrome;

[0010] (a5) Preparation of products for assessing or assisting in assessing the risk of death in patients with HFTS;

[0011] (a6) Preparation of products for monitoring or assisting in monitoring changes in the severity of the disease in patients with fever and thrombocytopenia syndrome;

[0012] (a7) Preparation of products for assessing or assisting in assessing the extent of tissue damage in patients with FEVER WITH THROMBOCYTOPIASIS.

[0013] Another object of the present invention is to provide a kit, the function of which is any one of the following (c1)-(c7):

[0014] (c1) Diagnosis or auxiliary diagnosis of fever with thrombocytopenia syndrome;

[0015] (c2) Screening or assisting in screening patients with HFTS (or screening or assisting in screening patients with HFTS who have a poor prognosis or screening or assisting in screening patients with HFTS who have a good prognosis);

[0016] (c3) To differentiate or assist in differentiating patients with HFTS who have a poor prognosis from those with a good prognosis;

[0017] (c4) To evaluate or assist in evaluating the prognosis and survival of patients with fever and thrombocytopenia syndrome;

[0018] (c5) To assess or assist in assessing the risk of death in patients with febrile thrombocytopenia syndrome;

[0019] (c6) Monitor or assist in monitoring changes in the severity of the disease in patients with fever and thrombocytopenia syndrome;

[0020] (c7) Assess or assist in the assessment of the extent of tissue damage in patients with febrile thrombocytopenia syndrome.

[0021] The kit provided by the invention comprises substances for detecting the content of iron ions.

[0022] In one embodiment of the present invention, the kit further comprises a data processing device A; the data processing device A comprises a data input module, a data recording module, a data comparison module and a conclusion output module;

[0023] The data input module is configured to input the iron ion content in the serum of the subject to be tested;

[0024] The data recording module is configured to store the iron ion content in the serum of the subject to be tested and the judgment threshold;

[0025] The data comparison module is configured to receive the iron ion content in the serum of the subject sent by the data input module, and call the judgment threshold from the data recording module for comparison with the iron ion content in the serum of the subject;

[0026] The conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine whether the subject to be tested is a patient with fever and thrombocytopenia syndrome according to a predetermined determination condition.

[0027] Furthermore, the predetermined judgment conditions are as follows: if the iron ion content in the serum of the subject to be tested is higher than the judgment threshold, the subject to be tested is or is suspected to be a patient with fever with thrombocytopenia syndrome; if the iron ion content in the serum of the subject to be tested is equal to or lower than the judgment threshold, the subject to be tested is or is suspected to be a patient with non-fever with thrombocytopenia syndrome.

[0028] The determination threshold can be obtained by referring to the method known in the technical field, and specifically, the iron ion content value (or the average iron ion content) in the serum of healthy people can be used as the determination threshold.

[0029] In another embodiment of the present invention, the kit further comprises a data processing device B; the data processing device B is composed of a data input module, a data recording module, a data comparison module and a conclusion output module;

[0030] The data input module is configured to input the iron ion content in the serum of the patient with fever and thrombocytopenia syndrome to be tested;

[0031] The data recording module is configured to store the iron ion content and judgment threshold in the serum of the patient with fever and thrombocytopenia syndrome to be tested;

[0032] The data comparison module is configured to receive the iron ion content in the serum of the patient with fever and thrombocytopenia syndrome to be tested sent by the data input module, and call the judgment threshold from the data recording module for comparison with the iron ion content in the serum of the patient with fever and thrombocytopenia syndrome to be tested;

[0033] The conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine the prognosis or death risk or degree of tissue damage of the patient with fever and thrombocytopenia syndrome to be tested according to predetermined determination conditions.

[0034] Furthermore, the predetermined judgment conditions are as follows: patients with fever and thrombocytopenia syndrome with high iron ion content have a poor prognosis, or patients with fever and thrombocytopenia syndrome with high iron ion content have a higher risk of death than patients with fever and thrombocytopenia syndrome with low iron ion content, or patients with fever and thrombocytopenia syndrome with high iron ion content have a higher degree of tissue damage than patients with fever and thrombocytopenia syndrome with low iron ion content.

[0035] The pyrexia and thrombocytopenia syndrome patients with high iron ion content are pyrexia and thrombocytopenia syndrome patients whose iron ion content is higher than the judgment threshold.

[0036] The fever with thrombocytopenia syndrome patients with low iron ion content are fever with thrombocytopenia syndrome patients whose iron ion content is equal to or lower than the judgment threshold.

[0037] The determination threshold can be obtained by referring to the methods known in the technical field, such as using the coxph function of the R package "survMisc" to analyze the iron ion content of each sample, and then selecting the iron ion content value corresponding to the smallest pvalue from the analysis result as the determination threshold; or the iron ion content of each sample can be arranged in order from low to high according to the median method, and then the iron ion content value that is in the center and divides the iron ion content into two equal parts is used as the determination threshold.

[0038] In any of the above-mentioned uses or kits, the unit of the iron ion content can be μg / L or μmol / L.

[0039] In any of the above-mentioned uses or kits, the substance for detecting the iron ion content is a substance for detecting the iron ion content in serum.

[0040] Furthermore, the substance for detecting the iron ion content in serum includes a reagent and / or an instrument for detecting the iron ion content in serum.

[0041] Furthermore, the reagent and / or instrument for detecting the iron ion content in serum is a reagent and / or instrument for detecting the iron ion content in serum by inductively coupled plasma mass spectrometry.

[0042] In one embodiment of the present invention, the iron ion content in serum can be detected as follows: a standard curve of trace element iron is prepared by inductively coupled plasma mass spectrometry, and the iron ion concentration value in the serum of the subject to be tested is obtained according to the standard curve.

[0043] In a specific embodiment of the present invention, the trace element iron is calibrated using the iron element coded GNM-SFE-002-2013 of the National Center for Standardization Materials Research.

[0044] In another embodiment of the present invention, an iron detection kit can be used to detect the iron ion content in serum.

[0045] In a specific embodiment of the present invention, the iron detection kit is a product of Mekang Biotechnology (cat. H309W).

[0046] In any of the above-mentioned applications or kits, the iron ion content (iron ion concentration) is the total content of trace element iron, including the content of divalent iron ions and the content of trivalent iron ions.

[0047] In any of the above applications or kits, the serum is derived from a blood sample of a subject to be tested, and is specifically prepared as follows: the blood sample of the subject to be tested is centrifuged, and the upper layer solution is aspirated to obtain the serum. The centrifugation condition may be 3000 rpm for 10 min.

[0048] In any of the above-mentioned applications or kits, the prognosis of survival includes prognosis of death and prognosis of survival.

[0049] The poor prognosis refers to a poor survival situation within one month, specifically a prognosis of death.

[0050] The good prognosis refers to a good survival condition within one month, specifically a prognosis of survival.

[0051] Any of the above-mentioned fever with thrombocytopenia syndrome is a disease caused by infection with fever with thrombocytopenia syndrome virus (new bunyavirus).

[0052] The present invention proves through experiments that the iron ion content in the serum of patients who survived the acute phase of fever with thrombocytopenia syndrome and patients who died in the acute phase is significantly higher than that of healthy controls, and the iron ion content in the serum of patients who died in the acute phase of fever with thrombocytopenia syndrome is significantly higher than that of patients who survived the acute phase, that is, the serum iron ion content is proportional to the severity of the fever with thrombocytopenia syndrome disease. The present invention first discovered that the iron ion content in the serum can effectively assist in the diagnosis of fever with thrombocytopenia syndrome, and at the same time has an important auxiliary guiding role in the prognosis of the development of the disease, which is not only convenient for doctors to make a diagnosis, but also helps to formulate a reasonable treatment plan based on the prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1The iron ion content in the serum of different types of patients. HC: healthy control; Recover.Sur: patients who survived the recovery period; Acute.Sur: patients who survived the acute period; Acute.Dea: patients who died in the acute period.

[0054] Figure 2 The relationship between serum iron ion concentration and onset time of different types of patients. SF.Sur: surviving patients; SF.Dea: dead patients.

[0055] Figure 3 This is a correlation analysis between the serum iron ion content and the lactate dehydrogenase LDH content and aspartate aminotransferase AST content of patients.

[0056] Figure 4 This is the ROC curve analysis of the iron ion content, lactate dehydrogenase LDH content and aspartate aminotransferase AST content in the patients' serum. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] Example 1: Analysis of serum iron content in patients with different types of SFTS

[0060] 1. Experimental Methods

[0061] 1. Test subjects

[0062] Subjects: SFTS patients who were laboratory confirmed and positive for SFTSV RNA by qRT-PCR were recruited from Xinyang Rongtong No. 154 Hospital in Xinyang City, Henan Province in 2019, including 26 patients who survived in the recovery period, 72 patients who survived in the acute period, and 41 patients who died in the acute period. Among them, patients who survived in the recovery period refer to SFTS patients whose admission time is greater than or equal to 7 days, whose infection signs are basically recovered, and whose outcome is survival (recorded as Acute.Sur), patients who survived in the acute period refer to SFTS patients whose admission time is less than 7 days, whose infection symptoms are obvious, and whose outcome is survival (recorded as Acute.Sur), and patients who died in the acute period refer to SFTS patients whose admission time is less than 7 days, whose infection symptoms are obvious, and whose outcome is death (recorded as Acute.Dea). All patients followed the treatment plan of the local hospital, and the outcome of death or survival refers to the outcome of death or survival from admission to the end of the investigation (the end time of the investigation is one month). Healthy people of the same period were included, with a total of 40 healthy controls (recorded as HC).

[0063] 2. Detection of iron ion content in serum

[0064] Blood samples were collected from the subjects using anticoagulant tubes (blood samples were collected from patients who survived the recovery period when the admission time was greater than or equal to 7 days, and blood samples were collected from patients who survived the acute period and patients who died in the acute period when the admission time was less than 7 days). The samples were centrifuged at 3000rpm for 10 minutes to collect serum. 0.2mL of serum was taken in a new centrifuge tube for sample testing. The iron ion content in the serum of the patients was determined by inductively coupled plasma mass spectrometry (ICP-MS), and the iron element GNM-SFE-002-2013 and blank controls were used for standardization to obtain the absolute value of the iron ion content in the serum of the subjects (in μg / L).

[0065] The specific detection method of ICP-MS includes the following steps: Use the iron element (dissolved in 2% dilute nitric acid solution) with the code number GNM-SFE-002-2013 of the National Center for Standard Materials Research for calibration. Prepare 0.2mL of serum sample, add it to 2% dilute nitric acid solution to a final volume of 3mL, and shake to mix. Prepare 2.8mL of sample diluent (2% dilute nitric acid solution), do not add any solution, and make a blank control. Detect on the machine according to the requirements of ICP-MS.

[0066] 2. Experimental Results

[0067] Test results such as Figure 1As shown, the results showed that the iron ion content in the serum of patients in the acute phase was significantly higher than that in healthy controls, the iron ion content in the serum of patients who died in the acute phase was significantly higher than that in patients who survived the acute phase, there was no significant difference in the iron ion content in the serum of patients in the recovery phase and healthy controls, and there was no significant difference in the iron ion content in the serum of patients who survived the acute phase and patients who survived the recovery phase.

[0068] The above results indicate that serum iron ion content can be used to distinguish the severity of fever with thrombocytopenia syndrome in diagnosis. Serum iron ion content is higher in the acute phase and even higher in patients who died.

[0069] Example 2: Changes in serum iron ion content in patients with different types of SFTS

[0070] 1. Experimental Methods

[0071] 1. Test subjects

[0072] Participants: A total of 818 SFTS patients were recruited from Xinyang No. 154 Hospital of Rongtong Xinyang City, Henan Province in 2022, who were confirmed by laboratory diagnosis and positive for SFTSV RNA by qRT-PCR, including 702 SFTS surviving patients and 116 SFTS dead patients. SFTS surviving patients refer to SFTS patients whose outcome was survival from admission to the end of the investigation (recorded as SF.Sur), and SFTS dead patients refer to SFTS patients whose outcome was death from admission to the end of the investigation (recorded as SF.Dea). Among them, the end time of the investigation is one month.

[0073] 2. Detection of iron ion content in serum

[0074] Blood samples were collected from each subject on different days after the onset of the disease (symptoms) using anticoagulant tubes, centrifuged at 3000rpm for 10 minutes, and serum was collected. 0.2mL of serum was taken into a new centrifuge tube for sample testing. The iron ion content in the patient's serum was measured using an iron detection kit (Meikang Biotechnology, cat. H309W), and the absolute value of the iron ion content in the subject's serum was calculated according to the instructions (in μmol / L).

[0075] The specific detection method of the iron detection kit includes the following steps: 30 μL of serum sample is mixed with 200 μL of reagent 1 and incubated for 3-5 minutes, and the absorbance value A1 is read, and 100 μL of reagent 2 is added, and the absorbance value A2 is read after incubation for 8 minutes. The serum iron ion content is calculated according to the calculation equation given in the kit.

[0076] 2. Experimental Results

[0077] Test results such as Figure 2As shown in the results, as the condition of SFTS patients progresses, the serum iron ion content will gradually increase. The increase in patients who died was more significant until death. The serum iron ion content of surviving patients first increased and then decreased. Compared with surviving patients, the serum iron ion content of deceased patients increased faster, and was significantly higher than that of surviving patients 6 days after the onset of symptoms. This shows that serum iron ions are related to the degree of improvement of the disease and are an effective marker for monitoring changes in the severity of fever with thrombocytopenia syndrome.

[0078] Example 3. Correlation analysis between iron ion content in patient serum and tissue damage index

[0079] 1. Experimental Methods

[0080] 1. Test subjects

[0081] The subjects were the same as those in Example 2.

[0082] 2. Detection of iron ion content in serum

[0083] The detection method is the same as in Example 2.

[0084] 3. Correlation analysis between iron ion content and lactate dehydrogenase LDH content and aspartate aminotransferase AST content

[0085] Calculate R using linear regression model 2 The correlation between iron ion content and lactate dehydrogenase LDH content or aspartate aminotransferase AST content was analyzed. Among them, the lactate dehydrogenase LDH content or aspartate aminotransferase AST content in the test subjects' serum was obtained by consulting the patient's medical records.

[0086] 2. Experimental Results

[0087] The analysis results are as follows Figure 3 The results showed that the iron ion content in the patient's serum was positively correlated with the lactate dehydrogenase LDH content in the serum. 2 =0.538, p<0.001. The serum iron ion content of patients was positively correlated with the serum aspartate aminotransferase AST content, R 2=0.459, p<0.001. The levels of lactate dehydrogenase (LDH) or aspartate aminotransferase (AST) were correlated with tissue damage (“Elevation of serum lactate dehydrogenase in AL amyloidosis reflects tissue damage and is an adverse prognostic marker in patients not eligible for stem cell transplantation.”, “Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation.” and “Markedly Elevated Aspartate Aminotransferase from Non-Hepatic Causes.”), indicating that serum iron ion content was positively correlated with tissue damage-related indicators. It can be seen that iron ion content has the potential to predict the degree of tissue damage in patients with fever and thrombocytopenia syndrome.

[0088] Example 4: ROC curve analysis of iron ion content, lactate dehydrogenase LDH content, and aspartate aminotransferase AST content in patient serum

[0089] 1. Experimental Methods

[0090] 1. Test subjects

[0091] The subjects were the same as those in Example 2.

[0092] 2. Detection of iron ion content in serum

[0093] The detection method is the same as in Example 2.

[0094] 3. ROC curve analysis of iron ion content, lactate dehydrogenase LDH content, and aspartate aminotransferase AST content

[0095] According to the serum iron ion content, lactate dehydrogenase LDH content or aspartate aminotransferase AST content in SFTS surviving patients and SFTS dead patients, ROC curve was drawn by cox regression model to evaluate the effectiveness of iron ion content, lactate dehydrogenase LDH content or aspartate aminotransferase AST content in predicting the outcome of SFTS patients. Among them, the serum lactate dehydrogenase LDH content and aspartate aminotransferase AST content of the subjects were obtained by reviewing the patient's medical records.

[0096] 2. Experimental Results

[0097] The results are as follows Figure 4 As shown, the results showed that in the early stage of viral infection, iron ions showed the strongest predictive ability ≤3 days and 4-6 days after the onset of symptoms, and iron ions still had a strong predictive ability 7-9 days after infection. The above results show that the iron ion content in serum has important value in predicting the fatal outcome of early SFTSV infection.

[0098] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a substance for detecting iron ion content in any one of the following (a1)-(a7): (a1) Preparation of products for diagnosis or auxiliary diagnosis of fever with thrombocytopenia syndrome; (a2) Preparation of products for screening or assisting in screening of patients with HFTS; (a3) Preparation of products that can differentiate or assist in differentiating patients with HFTS who have a poor prognosis from patients with HFTS who have a good prognosis; (a4) Preparation of products for evaluating or assisting in evaluating the prognosis and survival of patients with fever and thrombocytopenia syndrome; (a5) Preparation of products for assessing or assisting in assessing the risk of death in patients with HFTS; (a6) Preparation of products for monitoring or assisting in monitoring changes in the severity of the disease in patients with fever and thrombocytopenia syndrome; (a7) Preparation of products for assessing or assisting in assessing the extent of tissue damage in patients with FEVER WITH THROMBOCYTOPIASIS.

2. The use according to claim 1, Features: The substance for detecting the iron ion content is a substance for detecting the iron ion content in serum.

3. The use according to claim 2, Features: The substance for detecting the iron ion content in serum includes a reagent and / or an instrument for detecting the iron ion content in serum.

4. The use according to claim 3, Features: The unit of the iron ion content is μg / L or μmol / L.

5. The use according to any one of claims 1 to 4, Features: The iron ion content is the total content of trace element iron.

6. A kit comprising a substance for detecting iron ion content; the function of the kit is any one of the following (c1)-(c7): (c1) Diagnosis or auxiliary diagnosis of fever with thrombocytopenia syndrome; (c2) Screening or assisting in screening patients with fever and thrombocytopenia syndrome; (c3) To differentiate or assist in differentiating patients with HFTS who have a poor prognosis from those with a good prognosis; (c4) To evaluate or assist in evaluating the prognosis and survival of patients with fever and thrombocytopenia syndrome; (c5) To assess or assist in assessing the risk of death in patients with febrile thrombocytopenia syndrome; (c6) Monitor or assist in monitoring changes in the severity of the disease in patients with fever and thrombocytopenia syndrome; (c7) Assess or assist in the assessment of the extent of tissue damage in patients with febrile thrombocytopenia syndrome.

7. The kit according to claim 6, Features: The substance for detecting the iron ion content is a substance for detecting the iron ion content in serum.

8. The kit according to claim 7, Features: The substance for detecting the iron ion content in serum includes a reagent and / or an instrument for detecting the iron ion content in serum.

9. The kit according to claim 8, Features: The unit of the iron ion content is μg / L or μmol / L.

10. A kit according to any one of claims 6 to 9, Features: The iron ion content is the total content of trace element iron.

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